Mutation Research, 281 (1992) 133-135 133 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00

MUTLET 00621

No increase in chromosome aberrations in lymphocytes from workers exposed to nitrogen fertilisers

Armando Rojas Laboratorio de Mutagdnesis, Centro Nacional de lm,estigaciones Cientificas, Cubanacdn, Hacana (Cuba)

(Received 7 June 1991) (Revision received 24 September 1991)

(Accepted 7 October 1991)

Keywords: Genetic risk; Cytogenetic analysis; Nitrogen fertiliser industry

Summary

The putative genetic risk of people occupationally exposed to nitrogen fertilisers was studied using the structural chromosome aberration assay in peripheral blood lymphocytes. The exposed group included 23 subjects working at complex and mixed fertiliser plants. The percent of aberrant cells (Ab.C %) and break to cell ratio (B/C) were 0.95% and 0.01 respectively. The matched control group (20 subjects) was found to have 0.80% Ab.C and a B / C ratio of 0.0085. The results show a lack of detectable genetic damage in exposed people using this cytogenetic approach.

There is a considerable amount of experimen- tal evidence indicating that N-nitroso compounds are potent carcinogens (IARC, 1978). Such com- pounds can form by reaction of nitrosable amines or amides with nitrite. Endogenous formation of N-nitroso compounds can occur following inges- tion of large amounts of nitrate, and contact with nitrated sources. Another important, but not well understood, mechanism is via inhalation of nitro- gen oxides which has been correlated with excre- tion of N-nitrosodimethylamine in the urine (Tannenbaum, 1987).

Several authors have postulated a relationship between endogenous formation of N-nitroso

Correspondence: Dr. A. Rojas, Laboratorio de Mutag6nesis, Centro Nacional de Investigaciones Cientificas, Apartado 6880, Cubanac~n, Havana (Cuba).

compounds and increased risk for gastric cancer (Hill, 1979; Young, 1980). Specifically, different epidemiological studies have shown a positive correlation between gastric cancer and nitrogen fertiliser usage (Armijo and Coulson, 1975). How- ever, in some cases, no clear correlations have been shown concerning the aetiological role of these compounds in any human cancer (Fraser et al., 1980).

A large amount of evidence on endogenous synthesis of N-nitroso compounds demonstrated the excretion of nitroalkylamines in urine and even alkylated nucleic acid bases (Lu et al., 1987; Spiegelhalder et al., 1987; Shuker et al., 1987). However, genetic monitoring studies in humans are still rather scanty. Recently, Miller (1984) failed to show any effect of ingested nitrite on the level of unscheduled DNA synthesis in leukocytes from volunteers after consumption of amine and nitrite sources.

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Taking into account that nitrogen fertiliser workers are mainly exposed to nitrate, nitrogen oxides a n d / o r nitrated sources and the very scarce data concerning biological monitoring of such populations, a cytogenetic analysis was per- formed in order to evaluate the consequences of such exposure by studying structural chromosome aberrations.

and the break to cell ratio (B/C). The intergroup differences were statistically evaluated using Stu- dent's t-test.

Analysis of air samples was also done to mea- sure SO 2, NH 3 and NO 2 levels in the work-place air. Moreover, the level of total dust in air, mainly formed by fertiliser particulate aerosols, was also measured.

Materials and methods Results and discussion

Twenty=three workers at mixed and complex nitrogen fertiliser plants were selected, who had not been on drug treatment or had recent viral infections or X-rays during 3 months before ex- amination. The mean age was 33.2 + 5.8 years.

The control group comprised 20 subjects, with no known chemical exposure at work. The mean age was 36.5 _+ 6.1 years. The numbers of smokers in exposed and control groups represented 52% (12 out of 23) and 55% (11 out of 20), respec- tively.

The cytogenetic analysis was performed as fol- lows: blood samples were taken using heparinised syringes. Whole blood microcultures were done. Lymphocytes were grown in MEM (Gibco) sup- plemented with foetal calf serum 20% (Gibco) and stimulated for 50 h by phytohaemagglutinin (PHA 15, Wellcome).

All slides were coded and scored blind. A hundred metaphases were analysed for each sub- ject. A cell bearing at least a break or exchange was considered aberrant. Gaps, defined as un- stained regions with a length less than the width of one chromatid in the same chromosome, were also registered but not included for calculating the percentage of ceils with aberrations (Ab.C %)

Nitrogen fertiliser production involves expo- sure to various nitrate sources, so it may poten- tially lead to endogenous formation of N-nitroso compounds.

The results of the cytogenetic analysis, given in Table 1, failed to show any differences between exposed workers and control subjects.

All values obtained for SO 2, NH 3 and NO 2 were below the corresponding Cuban maximal allowable concentrations (MAC) of 20 m g / m 3, 20 m g / m 3, and 10 m g / m 3, respectively. On the other hand, the level of total dust (mainly formed by fertiliser particulate aerosols) reached values around MAC level (4 m g / m 3) and occasionally slightly higher values.

Several factors should be taken into considera- tion to explain our negative findings. Endogenous formation is limited by the pharmacokinetic pa- rameters of nitrate, its low conversion level into nitrite by oral microflora (Eisenbrand et al., 1980; Ellen et al., 1982) as well as the low pH values required or enzymatic catalysis by intestinal bac- teria at neutral pH (Archer et al., 1982). Also the nitrite level in the blood is maintained very low because nitrite is rapidly oxidised to nitrate by the action of oxyhaemoglobin (Rodney, 1976).

TABLE 1

RESULT OF THE CYTOGENETIC ANALYSIS

Group N Gaps B' B" E % AC ~' B / C ~'

Exposed 23 19 15 6 1 0.95 0.01 (0-3) (0-2) (0-1) - (0-2.0) (0-0.02)

Control 20 12 10 5 1 0.80 0.0085 (0-2) (0-2) (0-1) - (0-2.0) (0-0.02)

B', chromatid breaks; B", chromosome breaks; E, exchanges; % AC, percent of aberrant cells; B/C, break to cell ratio. " Calculated from individual means. Ranges are shown in parentheses.

Finally, 2 other aspects could have special rel- evance: the rapid metabolic decay of some N- nitroso compounds formed in vivo and the metabolic activation needed to transform them into active metabolites (Lijinsky, 1976). Though some nitroalkylamines reach to bloodstream, it seems that lymphocytes have not enough met- abolic activation capacity to reduce them to ac- tive metabolites, as shown in studies in vitro with per iphera l blood lymphocytes (Sankara- narayanan, 1981).

Taking into account that cytogenetic analysis failed to show any differences between exposed and control groups, and considering the very spe- cial features of the in vivo nitrosation process, further studies should be done, including analyti- cal procedures for determining N-nitroso com- pounds in body fluids and putative precursors in the work-place air. Besides these analytical ap- proaches, analysis of the mutagenicity of urine and work-place air using Ames test Salmonella strains will also be very convenient.

References

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Armijo, R., and A.H. Coulson (1975) Epidemiology of stom- ach cancer in Chile. The role of nitrogen fertilisers, Int. J. Epidemiol., 4, 301-309.

Eisenbrand, G., B. Spiegelhalder and R. Preussmann (1980) Nitrate and nitrate in saliva, Oncology, 37, 227-232.

Ellen, G., P.L. Schuller, E. Bujns, P.G.A.M. Froeling and H. Baadenhuijsen (1982) Volatile N-nitrosamines, nitrate and nitrite in urine and saliva of healthy volunteers after administration of large amounts of nitrate, in: H. Bartsch, I.K. O'Neill, M. Castegnaro and M. Okada (Eds.), N- Nitroso Compounds: Occurrence and Biological Effects, IARC Sci. Publ. No. 14, pp. 365-378.

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Miller, C.T. (1984) Unscheduled DNA synthesis in human leukocytes after a fish (amine source) meal with or without salad (nitrite source), in: I.K. O'Neill, R.C. von Borstel, C.T. Miller, J. Long and H. Bartsch (Eds.), N-Nitroso Compounds: Occurrence, Biological Effects and Rele- vance to Human Cancer, IARC Sci. Publ. No. 57, pp. 609-613.

Rodney, F.L. (1976) A mechanism for the conversion of oxyhemoglobin to methemoglobin by nitrite, Clin. Chem., 22, 1986-1990.

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Shuker, D.E.G., E. Bailey and P.B. Farmer (1987) Excretion of methylated nucleic acid bases as an indicator of expo- sure to nitrosable drugs, in: H. Bartsch, I. O'Neill and R. Schulte-Herman (Eds3, The Relevance of N-Nitroso Compounds to Human Cancer: Exposure and Mecha- nisms, IARC Sci. Publ. No. 84, pp. 407-410.

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Communicated by S.M. Galloway